Method for producing antithrombin-III, method for purifying it, and preparation containing it

Abstract

A method for producing antithrombin-III from an antithrombin-III-containing aqueous solution, comprising at least one of the following steps (a) and (b): (a) heating the antithrombin-III-containing aqueous solution in the presence of a stabilizing agent so that 85% or more of the activity of antithrombin-III before the heating is maintained after the heating, and that the ratio of an antithrombin-III monomer after the heating is maintained at 95% or more; and (b) treating the antithrombin-III-containing aqueous solution with a metal chelate resin and recovering a purified antithrombin-III.

What is claimed is: 
     
       1. A method for purifying antithrombin-III from an antithrombin-III-containing aqueous solution, comprising the following step: (a) heating the antithrombin-III-containing aqueous solution in the presence of, as a stabilizing agent, an organic acid or a salt thereof and a saccharide, optionally together with sodium chloride so that 85% or more of the activity of antithrombin-III before the heating is maintained after the heating, and that the ratio of an antithrombin-III monomer after the heating is maintained at 95% or more.   
     
     
       2. The method according to claim 1, wherein the organic acid or a salt thereof is selected from the group consisting of glycine, alanine, valine, leucine, isoleucine, oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid, maleic acid, fumaric acid, phthalic acid, aspartic acid, glutamic acid, malic acid, tartaric acid, citric acid and salts thereof. 
     
     
       3. The method according to claim 1, wherein the organic acid or a salt thereof is sodium citrate. 
     
     
       4. The method according to claim 1, wherein the saccharide is selected from the group consisting of glucose, fructose, galactose, mannose, arabinose, inositol, saccharose, lactose, maltose, mannitol, sorbitol, xylitol, glucosamine and N-acetyl-D-glucosamine. 
     
     
       5. The method according to claim 1, wherein the saccharide is sorbitol. 
     
     
       6. The method according to claim 1, wherein the organic acid or a salt thereof is contained in an amount of 3 to 20 w/v % in the solution. 
     
     
       7. The method according to claim 1, wherein the saccharide is contained in an amount of 10 to 60 w/v % in the solution. 
     
     
       8. The method according to claim 1, wherein the sodium chloride is contained in an amount of 0.5 to 3.0 mol/l in the solution. 
     
     
       9. The method according to claim 1, wherein the metal ion of the metal chelate resin is selected from the group consisting of a copper ion, a nickel ion and a cobalt ion. 
     
     
       10. The method according to claim 1, wherein the metal ion of the metal chelate resin is a copper ion. 
     
     
       11. The method according to claim 1, wherein the antithrombin-III-containing aqueous solution is subjected to a heat treatment before the step (b). 
     
     
       12. The method according to claim 12, wherein the heat treatment is carried out by heating the antithrombin-III-containing aqueous solution in the presence of, as a stabilizing agent, an organic acid or a salt thereof and a saccharide, optionally together with sodium chloride so that 85% or more of the activity of antithrombin-III before the heating is maintained after the heating, and that the ratio of an antithrombin-III monomer after the heating is maintained at 95% or more. 
     
     
       13. The method according to claim 12, wherein the antithrombin-III-containing aqueous solution is subjected to a dialysis treatment between the heat treatment and the step (a). 
     
     
       14. The method according to claim 1, wherein the heating is carried out at 50 to 70° C. for 10 minutes to 20 hours. 
     
     
       15. The method according to claim 1, wherein 95% or more of the activity of antithrombin-III before the heating is maintained after the heating. 
     
     
       16. The method according to claim 1, wherein the ratio of an antithrombin-III monomer after the heating is maintained at 99% or more. 
     
     
       17. The method according to claim 1, wherein the antithrombin-III-containing aqueous solution has a protein concentration of 0.1 to 5 w/v %. 
     
     
       18. A method for purifying antithrombin-III from an antithrombin-III-containing aqueous solution, comprising the following step: (a) contacting the antithrombin-III-containing aqueous solution with a metal chelate resin and recovering a purified antithrombin-III.   
     
     
       19. The method according to claim 18, wherein the antithrombin-III-containing aqueous solution has a protein concentration of 0.1 to 5 w/v %.